蔊菜幼苗抗菌核病及抗旱和耐湿特性的鉴定

以3个甘蓝型油菜(Brassica napus L.)品种‘中油821’(‘Zhongyou No.821’)、‘中双9号’(‘ZhongshuangNo.9’)及‘中油杂2号’(‘Zhongyouza No.2’)为对照,采用离体叶片菌丝块接种法、人工模拟干旱和湿害胁迫处理法对蔊菜〔Rorippa indica(L.)Hiern〕的抗菌核病〔Sclerotinia sclerotiorum(Lib.)de Bary〕、抗旱和耐湿特性进行了鉴定。结果表明:菌核病菌接种后,蔊菜幼苗叶片的病斑直径为1.75 cm,极显著小于3个甘蓝型油菜品种的病斑直径(3.25～3.60 cm)。经干旱胁迫后,3个油菜品种的幼苗严重萎蔫,茎粗、根长以及地上部分、根和全株的鲜质量和干质量均极显著低于对照;蔊菜幼苗轻度萎蔫,仅茎粗和根鲜质量分别极显著和显著低于对照,其他生长指标与对照差异不显著;蔊菜幼苗各生长指标的伤害指数均显著或极显著低于3个油菜品种。经湿害胁迫后,蔊菜和3个油菜品种幼苗的总叶片数和绿叶数较对照明显减少、黄叶数增加,但蔊菜幼苗的黄叶数显著少于3个油菜品种;3个油菜品种幼苗的茎粗、根长以及地上部分、根和全株的鲜质量和干质量总体上显著或极显著低于对照,而蔊菜幼苗仅茎粗、根长和根干质量显著低于对照,其他生长指标与对照差异不显著;蔊菜幼苗的茎粗,根长,地上部分、根和全株的鲜质量以及根和全株的干质量的伤害指数均显著或极显著低于3个油菜品种。研究结果显示:蔊菜对菌核病的抗性及抗旱和耐湿性均强于供试的3个甘蓝型油菜品种,是十字花科(Brassiaceae)中对菌核病抗性强、抗旱耐湿的优质基因源。     

<Emphasis Type="Italic">Brassica napus</Emphasis> possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> infection,wounding and defense hormone treatment

Most plants encode a limited set of polygalacturonase inhibitor (PGIP) genes that may be involved in aspects of plant development, but more importantly in the inactivation of polygalacturonases (PG) secreted by pathogens. Previously, we characterized two Brassica napus PGIP genes, BnPgip1 and BnPgip2, which were differentially expressed in response to pathogen infection and wounding. Here we report that the B. napus genome encodes a set of at least 16 PGIP genes that are similar to BnPgip1 or BnPgip2. This is the largest Pgip gene family reported to date. Comparison of the BnPGIPs revealed several sites within the xxLxLxx region of leucine rich repeats that form beta-sheets along the interacting face of the PGIP that are hypervariable and represent good candidates for generating PGIP diversity. Characterization of the regulatory regions and RT-PCR studies with gene-specific primers revealed that individual genes were differentially responsive to pathogen infection, mechanical wounding and signaling molecules. Many of the BnPgip genes responded to infection by the necrotic pathogen, Sclerotinia sclerotiorum; however, these genes were also induced either by jasmonic acid, wounding and salicylic acid or some combination thereof. The large number of PGIPs and the differential manner in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests.     

Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris.

The spectrum of resistance to isolates of Leptosphaeria maculans and the map location of a new blackleg resistance gene found in the canola cultivar Brassica napus 'Surpass 400' are described. Two blackleg resistance genes, LepR1 and LepR2, from B. rapa subsp. sylvestris and introgressed in B. napus were identified previously. 'Surpass 400' also has blackleg resistance introgressed from B. rapa subsp. sylvestris. Using 31 diverse isolates of L. maculans, the disease reaction of 'Surpass 400' was compared with those of the resistant breeding lines AD9 (which contains LepR1), AD49 (which contains LepR2), and MC1-8 (which contains both LepR1 and LepR2). The disease reaction on 'Surpass 400' was different from those observed on AD9 and MC1-8, indicating that 'Surpass 400' carries neither LepR1 nor both LepR1 and LepR2 in combination. Disease reactions of 'Surpass 400' to most of the isolates tested were indistinguishable from those of AD49, which suggested 'Surpass 400' might contain LepR2 or a similar resistance gene. Classical genetic analysis of F1 and BC1 plants showed that a dominant allele conferred resistance to isolates of L. maculans in 'Surpass 400'. The resistance gene, which mapped to B. napus linkage group N10 in an interval of 2.9 cM flanked by microsatellite markers sR12281a and sN2428Rb and 11.7 cM below LepR2, was designated LepR3. A 9 cM region of the B. napus genome containing LepR3 was found to be syntenic with a segment of Arabidopsis chromosome 5.     

Resistance of some cultivated Brassicaceae to infestations by Plutella xylostella (Lepidoptera: Plutellidae)

Selecting insect-resistant plant varieties is a key component of integrated management programs of oligophagous pests such as diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), but rigorous research on important life history parameters of P. xylostella in relation to host plant resistance is rare. We evaluated six conventional brassicaceous species, namely, Brassica napus L. 'Q2', B. rapa L., B. juncea (L.) Czern., B. carinata L., B. oleracea L., and Sinapis alba L., and two herbicide-tolerant cultivars, namely, B. napus 'Liberty' and B. napus 'Conquest' for their resistance against P. xylostella. Brassicaceae species and cultivars varied considerably in their susceptibilities as hosts for P. xylostella. Sinapis alba and B. rapa plants were highly preferred by ovipositing females and trichome density on adaxial and abaxial leaf surfaces had nonsignificant effects on P. xylostella oviposition. Larval survival was similar on the genotypes we tested, but host plants significantly affected larval and pupal developmental time, herbivory, pupal weight, silk weight, adult body weight, forewing area and longevity (without food) of both male and female P. xylostella. Larval and pupal development of females was fastest on B. juncea and S. alba, respectively. Specimens reared on B. napus Liberty and B. oleracea, respectively, produced the lightest female and male pupae. Defoliation by both female and male larvae was highest on B. rapa, whereas least herbivory occurred on S. alba. Females reared on S. alba were heavier and lived longer in the absence of food than their counterparts raised on other tested host plants. Brassica oleracea could not compensate for larval feeding to the level of the other species we evaluated. B. napus Conquest, B. napus Q2, B. carinata, B. rapa, and S. alba produced, respectively, 1.6-, 1.8-, 1.8-, 3.9-, and 5.5-fold heavier root systems when infested than their uninfested counterparts, suggesting that these species were better able to tolerate P. xylostella infestations.     

Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot

Canola is an important agricultural crop imparting a significant contribution to global oilseed production. As such, optimizing yield and quality is of paramount importance and canola production can be significantly affected by sclerotinia stem rot. The utility of recombinant antibody technology in plant protection has been explored by many researchers and shows promise for the generation of new lines of agriculturally significant crops with greater resistance to diseases. The objective of the current study was to generate recombinant pathogen specific antibody (scFv)-expressing transgenic Brassica napus plants with increased tolerance to the phytopathogenic fungus, Sclerotinia sclerotiorum. Transgenic canola (B. napus) lines expressing S. sclerotiorum-specific scFv antibody showed a significant level of tolerance towards S. sclerotiorum as compared to their non-transformed counterparts. Both incidence and progression of S. sclerotiorum-induced disease symptoms were reduced in plants expressing the recombinant scFv.     

The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea

BACKGROUND: Plants have evolved efficient mechanisms to combat pathogen attack. One of the earliest responses to attempted pathogen attack is the generation of oxidative burst that can trigger hypersensitive cell death. This is called the hypersensitive response (HR) and is considered to be a major element of plant disease resistance. The HR is thought to deprive the pathogens of a supply of food and confine them to initial infection site. Necrotrophic pathogens, such as the fungi Botrytis cinerea and Sclerotinia sclerotiorum, however, can utilize dead tissue. RESULTS: Inoculation of B. cinerea induced an oxidative burst and hypersensitive cell death in Arabidopsis. The degree of B. cinerea and S. sclerotiorum pathogenicity was directly dependent on the level of generation and accumulation of superoxide or hydrogen peroxide. Plant cells exhibited markers of HR death, such as nuclear condensation and induction of the HR-specific gene HSR203J. Growth of B. cinerea was suppressed in the HR-deficient mutant dnd1, and enhanced by HR caused by simultaneous infection with an avirulent strain of the bacterium Pseudomonas syringae. HR had an opposite (inhibitory) effect on a virulent (biotrophic) strain of P. syringae. Moreover, H(2)O(2) levels during HR correlated positively with B. cinerea growth but negatively with growth of virulent P. syringae. CONCLUSIONS: We show that, although hypersensitive cell death is efficient against biotrophic pathogens, it does not protect plants against infection by the necrotrophic pathogens B. cinerea and S. sclerotiorum. By contrast, B. cinerea triggers HR, which facilitates its colonization of plants. Hence, these fungi can exploit a host defense mechanism for their pathogenicity.     

<Emphasis Type="Italic">Germin-like protein</Emphasis> gene family of a moss, <Emphasis Type="Italic">Physcomitrella patens</Emphasis>, phylogenetically falls into two characteristic new clades

We identified 77 EST clones encoding germin-like proteins (GLPs) from a moss, Physcomitrella patens in a database search. These Physcomitrella GLPs (PpGLPs) were separated into seven groups based on DNA sequence homology. Phylogenetic analysis showed that these groups were divided into two novel clades clearly distinguishable from higher plant germins and GLPs, named bryophyte subfamilies 1 and 2. PpGLPs belonging to bryophyte subfamilies 1 lacked two cysteines at the conserved positions observed in higher plant germins or GLPs. PpGLPs belonging to bryophyte subfamily 2 contained two cysteines as observed in higher plant germins and GLPs. In bryophyte subfamily 1, 12 amino acids, in which one of two cysteines is included, were deleted between boxes A and B. Further, we determined the genomic structure of all of seven PpGLP genes. The sequences of PpGLPs of bryophyte subfamily 1 contained one or two introns, whereas those of bryophyte subfamily 2 contained no introns. Other GLPs from bryophytes, a liverwort GLP from Marchantia polymorpha, and two moss GLPs from Barbula unguiculata and Ceratodon purpureus also fell into bryophyte subfamily 1 and bryophyte subfamily 2, respectively. No higher plant germins and GLPs were grouped into the bryophyte subfamilies 1 and 2 by our analysis. Moreover, we revealed that PpGLP6 had manganese-containing extracellular superoxide dismutase activity. These results indicated that bryophyte possess characteristic GLPs, which phylogenetically are clearly distinguishable from higher plant GLPs.     

Identification of prior candidate genes for Sclerotinia local resistance in Brassica napus using Arabidopsis cDNA microarray and Brassica-Arabidopsis comparative mapping

Arabidopsis was the leading model of dicot plant. An accomplished platform had been established for functional genomics studies. The platform had largely facilitated molecular biology research of Arabidopsis itself as well as research on its phylogenetic related plants[1,2]. Brassica napus, as an important cooking oil crop, had a close phylogenetic relationship with Arabidopsis[3―5]. In order to take advantage of avail-able Arabidopsis genetic and molecular tools, Girke et al. had explored …     

Germins and germin like proteins: an overview

Molecular investigations during wheat germination have revealed unique developmentally regulated proteins, designated as germins, which show remarkable resistance to broad specificity proteases and to dissociation in SDS. Germins in cereals have an oxalate oxidase activity, which generates H2O2 from the oxidative breakdown of oxalate thereby playing a significant role in plant development and defense. Germin like proteins (GLPs) exhibit sequence and structural similarity with the cereal germins but mostly lack oxalate oxidase activity. Germins and germin like proteins (GLPs) are a class of developmentally regulated glycoproteins characterized by a beta-barrel core structure, a signal peptide, and are associated with the cell wall. GLPs exhibit a broad range of diversity in their occurrence and activity in organisms ranging from myxomycetes, bryophytes, pteridophytes, gymnosperms and angiosperms. Germins and GLPs are thought to play a significant role during zygotic and somatic embryogenesis (wheat and Pinus, respectively), salt stress (barley and Mesembryanthemum crystallinum), pathogen elicitation (wheat and barley), and heavy metal stress, etc. Characterization and cloning of some of the genes encoding germins and GLPs has facilitated a better understanding of their regulation and raised their potential of biotechnological application.     

Yield reduction in Brassica napus, B. rapa, B. juncea, and Sinapis alba caused by flea beetle (Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae)) infestation in northern Idaho

Phyllotreta cruciferae is an important insect pest of spring-planted Brassica crops, especially during the seedling stage. To determine the effect of early season P. cruciferae infestation on seed yield, 10 genotypes from each of two canola species (Brassica napus L. and Brassica rapa L.) and two mustard species (Brassica juncea L. and Sinapis alba L.) were grown in 2 yr under three different P. cruciferae treatments: (1) no insecticide control; (2) foliar applications of endosulfan; and (3) carbofuran with seed at planting plus foliar application of carbaryl. Averaged over 10 genotypes, B. rapa showed most visible P. cruciferae injury and showed greatest yield reduction without insecticide application. Mustard species (S. alba and B. juncea) showed least visible injury and higher yield without insecticide compared with canola species (B. napus and B. rapa). Indeed, average seed yield of S. alba without insecticide was higher than either B. napus or B. rapa with most effective P. cruciferae control. Significant variation occurred within each species. A number of lines from B. napus, B. juncea, anid S. alba showed less feeding injury and yield reduction as a result of P. cruciferae infestation compared with other lines from the same species examined, thus having potential genetic background for developing resistant cultivars.     

Comparative mapping reveals similar linkage of functional genes to QTL of yield-related traits between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Oryza sativa and Brassica napus-two important crops for food and oil, respectively-share high seed yield as a common breeding goal. As a model plant, O. sativa genomics have been intensively investigated and its agronomic traits have been advanced. In the present study, we used the available information on O. sativa to conduct comparative mapping between O. sativa and B. napus, with the aim of advancing research on seed-yield and yield-related traits in B. napus. Firstly, functional markers (from 55 differentially expressed genes between a hybrid and its parents) were used to detect B. napus genes that co-localized with yield-related traits in an F(2:3) population. Referring to publicly available sequences of 55 B. napus genes, 53 homologous O. sativa genes were subsequently detected by screening, and their chromosomal locations were determined using silico mapping. Comparative location of yield-related QTL between the two species showed that a total of 37 O. sativa and B. napus homologues were located in similar yield-related QTL between species. Our results indicate that homologous genes between O. sativa and B. napus may have consistent function and control similar traits, which may be helpful for agronomic gene characterization in B. napus based on what is known in O. sativa.     

Oxalic acid, a pathogenicity factor for Sclerotinia sclerotiorum, suppresses the oxidative burst of the host plant

Effective pathogenesis by the fungus Sclerotinia sclerotiorum requires the secretion of oxalic acid. Studies were conducted to determine whether oxalate aids pathogen compatibility by modulating the oxidative burst of the host plant. Inoculation of tobacco leaves with an oxalate-deficient nonpathogenic mutant of S. sclerotiorum induced measurable oxidant biosynthesis, but inoculation with an oxalate-secreting strain did not. Oxalate inhibited production of H(2)O(2) in tobacco and soybean cultured cell lines with a median inhibitory concentration of approximately 4 to 5 mM, a concentration less than that measured in preparations of the virulent fungus. Several observations also indicate that the inhibitory effects of oxalate are largely independent of both its acidity and its affinity for Ca(2)+. These and other data demonstrate that oxalate may inhibit a signaling step positioned upstream of oxidase assembly/activation but downstream of Ca(2)+ fluxes into the plant cell cytosol.     

导入双价基因的转基因杂交油菜亲本及其对菌核病抗性的研究

比较了乙酰丁香酮、pH、共培养温度及不同激素配比对根癌农杆菌转化油菜(Brassica napus)的影响, 建立了油菜高效转化体系。按该体系, 将组成型表达几丁质酶和β-1, 3-葡聚糖酶基因转化甘蓝型双低杂交油菜亲本恢复系和保持系, 获得了转基因恢复系和保持系植株。对再生植株进行PCR和Southern Blot检测, 结果表明外源基因已整合到油菜基因组中。连续3代的PCR检测及转基因植株抗病性在自交后代中得到保持, 证实外源基因已遗传到T3代。对转基因植株T1代离体叶进行菌核病菌丝体接种和T1及T2代大田自然侵染鉴定, 结果表明, 转基因恢复系棚40-12、棚40-32和保持系96B-2对菌核病的抗性比受体对照大幅度提高, 大田鉴定其发病率连续2年均比受体对照减少78%以上, 比抗病品种‘中油821’减少75%以上, 病情指数比受体对照和‘中油821’减少均达显著水平, 其抗病性能在后代稳定遗传, 获得了高抗菌核病的转基因育种材料。